The piglet feed was supplemented with 0, 4, and 8% black soldier fly insect powder (C group, T1 group, T2 group). The feed for all groups was prepared in accordance with NRC (2012) standards.

For this study, 12 weaned piglets with an initial average weight of 7.68 ± 0.26 kg were randomly categorized into C group (K88 group), T1 group (4% H. illucens larvae + K88 group), and T2 group (8% H. illucens larvae + K88 group) with four replicates in each group. The feed for all groups was prepared in accordance with NRC (2012) standards. Piglets were fed on a diet supplemented with 0, 4, and 8% black soldier fly insect powder (C group, T1 group, T2 group) for 28 days. On the 29th day, the ETEC K88 (50 × 10⁹ CFU/mL) was infused in the piglets using an intragastric catheter (14, 15). On the 32nd day, 10 mL of blood from the anterior vena cava of the piglets was collected in tubes containing an anticoagulant and used for metabolite detection. Ileum tissue was taken and placed in PBS buffer to wash away the contents, divided into two portions. One portion was stored in 4% formaldehyde solution for sectioning, and from the other portion, the intestinal mucosa was scraped for detecting related protein expression.

For examining the microbial diversity, the Illumina HiSeq sequencing platform was used to construct a small fragment library and sequenced, followed by diversity analysis, difference analysis, correlation analysis, and function prediction analysis as previously described (16).

To Detect the Effect of H. illucens on Metabolites in Serum of Weaned Piglets, GC-MS was performed to detect metabolites such as organic acids, fatty acids, and indole in serum (17, 18).

In this study, male mice born in the same litter and aged 6–8 weeks were used after procuring them from Zhuhai Bestone Biotechnology Co., Ltd. The mice were kept in an environment with freely available feed, natural light, and temperature at 22–25°C. To establish an ETEC infection model, Allen's method (19) was referred. In brief, water (5 g/L) supplemented with streptomycin was given to all mice for 72 h before ETEC infection to clear the natural flora in the intestines. Twelve hours before ETEC inoculation, the streptomycin-treated water was replaced with sterile water, and 1 × 10⁹ CFU of ETEC strain K88 was diluted with 0.1 M carbonate buffer, pH 9.0, and given to mice by gavage (20). The control group was given an equal volume of sterile phosphate-buffered saline (PBS). Two weeks before drinking streptomycin, the mice were given citric acid (5 g/L) by adding to the drinking water until the end of the experiment (21). Five days after ETEC infection, the animals were sacrificed to collect serum, spleen, and ileum and stored in a refrigerator at −80°C.

IPI-2I cells and RAW264.7 macrophages, purchased from BeiNa Culture Collection (Beijing, China), were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (Clark, Australia) and were maintained at 37°C in a humidified chamber of 5% CO₂.

In this study, RAW264.7 cells were categorized into four groups. The control group was cultured with D-MEM/F-12 containing 10% fetal bovine serum. The LPS group was treated with 1 μg/mL LPS for 24 h. The CA group was treated with 4 mmol/L citric acid for 24 h. The CA pretreatment group (CA+LPS group) was pre-protected with 4 mmol/L citric acid for 1 h and then treated with 1 μg/mL LPS for 24 h. The treatment of IPI-2I cells was consistent with that of RAW264.7 cells. The Cell Counting Kit-8 assay estimated the optimal concentration of lipopolysaccharides (LPS) and CA to be 1 μg/mL and 4 mmol/L, respectively.

Total RNA was extracted from the ileum tissue, RAW264.7 macrophages, primary splenic cells, and IPI-2I cells using TRIzol (Invitrogen, Carlsbad, CA), and cDNA was generated using a commercial RT-PCR kit (Takara Shuzo Co., Ltd., Kyoto, Japan). Then, real-time PCR was conducted using the SYBR Green QuantiTect RT-PCR kit (Roche, South San Francisco, CA), and each sample was analyzed in triplicate. The primer sequences for mice and Sus scrofa are shown in Tables 1, 2.

Total proteins were extracted from the ileum tissue and IPI-2I cells following the procedure described previously (22). Subsequently, western blotting was performed following the standard protocol (22). The antibodies used were: Occludin (1:1,000; EPR8208, Abcam), Bax (1:5,000; 50599-2-lg, Proteintech), Bcl-2 (1:1,000; 12789–1-AP, Proteintech), β-Actin (1:1,000; 4970, CST), HRP Conjugated AffiniPure Goat Anti-Rabbit IgG (1:5,000; BA1055, BOSTER).

Elisa kits (MEIMIAN, MM-0057M1, MM-0055M1, MM-45051M1, and MM-44850M1) were used. Detection of IgA and IgG in piglet serum, IgA, IgG, B cell-activating factor (BAFF), TGF-β, and IL-10 in mouse serum and BAFF, TGF-β, and IL-10 in RAW264.7 cell culture medium. Specific steps were performed following instructions from the manufacturer.

Mouse primary splenocyte suspension was collected and stained with combinations of the following antibodies: BV421 anti-IgG, AF750 anti-CD19 (Biolegend), PE anti-CD138 (Syndecan-1, R&D Systems), and FITC anti-IgA (Southern Biotech). For intracellular detection of IgG and IgA, Golgi-stop was added to cell cultures 4 h prior to harvest and extracellular staining was performed using anti-CD19 and anti-CD138 Abs. Cells were then fixed and permeabilized before intracellular staining with anti-IgG and anti-IgA Abs.Stained cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA).

The ileum tissues of piglets and mice were taken, and conventional slices were prepared. One was used for HE staining to observe the pathological changes of the tissue under a light microscope, and the other was used for TUNEL staining to observe the apoptosis ratio in the tissues, strictly following the kit instructions.

RAW264.7 cells were centrifuged and seeded in 96-well plates at a density of 2 × 10⁴ cells/well, with five repetitions for each treatment group. Twenty-four hours after seeding the cells, the medium was changed to DMEM basal medium. After 4 h of treatment, citric acid was added for pre-protection, and then 2 h later LPS was added for stimulation. After 24 h of stimulation, the medium was discarded, and 100 μL of CCK8 premix solution was added to each well. Finally, after 1 h, the absorbance was detected at 460 nm.

The results are expressed as mean ± SEM. Statistical analysis was performed using Student's t-test or one-way analysis of variance (ANOVA) and analyzed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA). The P value < 0.05 was considered statistically significant.

After ETEC infection, the serum IgA and IgG levels of piglets in the H. illucens larvae feed groups were significantly higher than those in the C group (Figure 1A). HE staining results showed that the ileum Vh/Cd (villus height/crypt depth) ratio of the H. illucens larvae feed groups was significantly higher than that of the C group (Figure 1B). The gene expression of the inflammatory factors IL-6, TNF-α, IL-1β, and tight junction protein ZO-1 in the ileal mucosa was also detected (Figure 1C). The expression of TNF-α and IL-1β in the H. illucens larvae feed groups decreased significantly, but the expression of ZO-1 increased significantly. Concomitantly, the results of TUNEL staining showed that compared with the C group, ETEC K88-mediated apoptosis was significantly inhibited in the H. illucens larvae feed groups (Figure 1D). The changes in the ratio of Bcl-2/Bax were detected by western blotting, and the results reveal significantly increased anti-apoptotic Bcl-2/Bax ratio of the H. illucens larvae feed groups (Figure 1E). The above results indicate that under the challenge of ETEC K88, H. illucens larvae feed not only improved the immune performance but also attenuated the damage caused by ETEC K88 by reducing apoptosis and inflammation, and enhancing the integrity of the intestinal barrier.

In order to reveal the specific mechanism in piglets under H. illucens larvae supplement, metabonomic analyses were carried out on the serum of the three groups of piglets. We found that with the increase in the content of H. illucens larvae, the citric acid content in the serum (Figure 2A), which was confirmed to be related to inflammation and immunity, increased in a dose-dependent manner. Therefore, we suppose that H. illucens larvae might affect the immune performance and intestinal health of piglets through citric acid derived from it.

In order to confirm the relationship between the CA derived from H. illucens larvae and the immune performance of piglets, mouse peritoneal macrophages RAW264.7 cells were used to study the immune regulation mechanism of citric acid in vitro. On analyzing gene expression of non-specific immune-related cytokines IL-6, TNF-α, and IL-1β, we observed a significant increase in the expression of non-specific immune-related cytokines IL-6, TNF-α, and IL-1β after LPS challenge (Figure 3A). Compared with the LPS group, the LPS+CA group exhibited a significantly attenuated increase in the expression of IL-6, TNF-α, and IL-1β (Figure 3A). The above results showed that the treatment of citric acid can significantly inhibit the excessive production of inflammatory factors IL-6, TNF-α, and IL-1β in RAW264.7 cells induced by LPS.

The expression of specific immune-related cytokines, such as BAFF, TGF-β, and IL-10, indicated that citric acid treatment reduced the expression of BAFF significantly; however, that of TGF-β and IL-10 increased significantly (Figure 3B). In addition, compared with the NT group, the CA group exhibited significantly increased expression of BAFF and decreased expression of TGF-β, while an increase in IL-10 level was also observed (Figure 3B). Likewise, we also assessed the secretion of specific immune-related cytokines by macrophages, and the results were consistent with their mRNA levels (Figure 3C). The above results showed that in the case of LPS challenge, the addition of citric acid could promote the secretion of BAFF, a B cell-activating factor by macrophages, and concurrently reduce the release of TGF-β, which promotes the activation and proliferation of B cells. Therefore, we preliminarily speculated that CA might be related to the B lymphocyte proliferation or differentiation.

To further verify the relationship between CA and B cell proliferation, primary mouse spleen cells were extracted and cultured. For T lymphocyte proliferation, ConA was used as a positive control, and for B lymphocyte proliferation, LPS was used as a positive control. CCK8 assay at 24 h showed a significant decrease in cell viability in the CA group compared with that of the NT group (Figure 4A). At the same time, the cell viability of the ConA+CA group was significantly lower than that of the ConA group, and a similar trend was observed for the LPS+CA group and the LPS group (Figure 4A). The result of CCK8 at 48 h was consistent with that at 24 h, further confirming that citric acid inhibited B cell proliferation (Figure 4A).

To verify the effect of citric acid on B cell activation, C57BL/6 mice were categorized into NT group, ETEC group, CA group, and CA+ETEC group. Flow cytometry of primary spleen cells of mice revealed that ETEC infection significantly reduced percentage of the IgG⁺ cells among CD19⁺ B cells, and a similar trend was observed for IgA⁺ B cells (Figure 4B). Compared with the NT group, citric acid significantly increased the IgG⁺ and IgA⁺ cell ratio in CD138⁺ CD19⁺ cells (Figure 4B). At the same time, citric acid significantly reversed the decrease in IgG⁺ and IgA⁺ population in CD138⁺CD19⁺ cells caused by ETEC infection (Figure 4B). To further verify results, the levels of IgA and IgG in the serum of mice were tested, and the results were consistent with those of flow cytometry (Figure 4C). Estimation of BAFF, TGF-β, and IL-10 levels in the serum of mice showed that upon ETEC infection, the contents of BAFF in the serum of mice reduced significantly, the addition of citric acid significantly alleviates BAFF reduction compared with that in the ETEC group (Figure 4D). The level of IL-10 was consistent with that of BAFF (Figure 4D). Likewise, the TGF-β level increased significantly after ETEC infection, citric acid attenuates the increased TGF-β (Figure 4D). The above experimental results showed that citric acid could stimulate macrophages to secrete a large amount of cytokines that promoted B cell activation, thereby increasing IgA and IgG production. These results were also found consist with increased IgA and IgG levels in the serum of piglets in the H. illucens larvae feed group.

To verify the effect of CA on intestinal epithelial integrity, the porcine ileal epithelial cell line IPI-2I was cultured and divided into NT, CA, LPS, and LPS+CA groups. Assessment of the expression of genes coding for inflammatory cytokine IL-6, tight junction protein ZO-1, and apoptosis-related proteins Bcl-2 and Bax, and tight junction protein Ocln showed that after LPS stimulation, the mRNA level of IL-6 increased significantly, while that of ZO-1 decreased significantly, and the level of Ocln protein decreased significantly (Figures 5A–C). Concurrently, the anti-apoptotic ratio of Bcl-2/Bax also decreased significantly (Figures 5B,C). Compared with the NT group, the CA group revealed significantly increased expression of tight junction proteins ZO-1 and Ocln, increased anti-apoptotic ratio of Bcl-2/Bax, and decreased expression of the inflammatory cytokine IL-6 (Figures 5A–C). These results indicate that citric acid inhibited the release of IL-6, promoted the expression of intestinal tight junction protein, and led to an increase in the anti-apoptotic ratio of cells, thus maintaining intestinal integrity.

Since citric acid has a protective effect on porcine ileal epithelial cells, the protective effects of citric acid were evaluated in an ETEC infected mice model. HE staining showed that after ETEC K88 infection, the ileal villi were sparse and messy, while those in the CA group were denser and more regular (Figure 6A). Compared with the ETEC group, the addition of citric acid made the ileal villi more compact and regular (Figure 6A). At the same time, after ETEC K88 infection, the expression of TNF-α in the jejunum increased significantly, that of IL-10 decreased significantly, and the expression of Cldn5 and Ocln also decreased significantly (Figure 6B). Compared with the NT group, the CA group exhibited significantly reduced expression of inflammatory factor TNF-α and promoted the expression of the anti-inflammatory cytokine IL-10 and the tight junction proteins Cldn5 and Ocln (Figure 6B). Compared with the ETEC group, the addition of citric acid inhibited the increase in TNF-α expression and the decrease in the expression of tight junction proteins caused by ETEC infection (Figure 6B).

TUNEL results showed a significantly increased percentage of apoptotic area in the ETEC group, and there was no difference between the CA group and the NT group (Figure 6C). Compared with the ETEC group, the addition of citric acid significantly reduced the percentage of the apoptotic area (Figure 6C) and increased the anti-apoptotic ratio of Bcl-2/Bax (Figure 6D).

Based on the above results, we speculated that citric acid production might be metabolized by intestinal microbes. Combined analysis of fecal 16S sequencing results and serum metabolomics showed a positive correlation of citric acid with intestinal Massilia (Figure 7A). In order to verify whether Massilia produced citric acid, it was cultured in R2A liquid medium for 24 h in vitro, and the content of citric acid in the medium was detected and compared with the pure R2A liquid medium. We observed that compared with the control group, the citric acid content in the culture medium of the Massilia group was almost doubled (Figure 7B).

Therefore, we could show that H. illucens larvae feed increased the content of Massilia in the intestine, whose metabolite—citric acid—improved the poor immune performance and intestinal barrier damage of piglets caused due to ETEC K88 infection.